Method for treatment of diseases with dioxoanthracene sulphonate derivatives

ABSTRACT

Compounds that may have anti-inflammatory activity have the general formula (I): Wherein R 1 , R 2 , R 3  are each independently H or a C 1-4  alkyl group or a C 2-4  acyl group; R 4  and R 5  are each independently H or a group of formula —SO 3 R 6 , wherein R 6  is H or a C 1-4  alkyl group or a C 2-4  acyl group; with the proviso that at least one of R 4  and R 5  is a group of formula —SO 3 R 6 , or a pharmaceutically acceptable salt thereof.

The present invention relates to certain dioxoanthracene sulphonatederivatives, to a process for the preparation thereof, and to the use ofthe compound as a medicament, particularly in the therapy of conditionsthat are influenced by pro-inflammatory cytokines of the IL-1 family,particularly inflammatory and auto-immune diseases, for instancearthritic diseases.

Rhein, 4,5-dihydroxy-9,10-dioxo-2-anthracene carboxylic acid, and itsdiacetylated derivative diacerein, are known for a number ofpharmaceutical applications. Particularly rhein and diacerein are knownfor use in the treatment of arthritic diseases, in particularosteoarthritis and rheumatoid arthritis, for instance as described inU.S. Pat. No. 4,244,968, GB 1 578 452, EP 544 880 B1, EP 636 602 B1 andU.S. Pat. No. 6,610,750, and psoriasis and associated conditions, asdescribed in EP 1 248 608 B1 Rhein and diacerein have also beendescribed for the treatment of various conditions, for instanceinflammatory diseases, auto-immune diseases, vascular diseases, painrelief, diabetic nephrosis.

The cytokines IL-1 (α, β) and TNF-α are considered to play an essentialrole in the mediation of the inflammatory process and cartilagedegradation. IL-1 and TNF-α are also considered to be implicated in themediation of biological responses to endotoxins and other infectiousstimuli. An extensive review of pro-inflammatory and anti-inflammatorycytokines is given by C. A. Dinarello, MD et L. L. Moldawer, PhD in theprimer for clinicians “Proinflammatory and Anti-inflammatory Cytokinesin Rheumatoid Arthritis”, 2000, Amgen Inc. The cytokines IL-1 and TNF-αhave been implicated in the mechanism of a number of inflammatory andauto-immune conditions such as osteoarthritis, rheumatoid arthritis,psoriatic arthritis, psoriasis, Paget's disease, osteoporosis,inflammatory bowl diseases including ulcerative colitis and Crohn'sdisease, endometriosis, Wegener's granulomatosis, neurologicaldysfunctions such as Alzheimer's disease and Parkinson's disease,myeloma, myeloid leukaemia, bone metastasis, diabetic nephrosis, chronicheart disease, arthrosclerosis, asthma.

Diacerein and its active metabolite rhein are known to inhibit thesynthesis and activity of pro-inflammatory catabolic cytokines of theinterleukin-1 (IL-1) family, particularly IL-1β. Rhein and diacereinhave been shown to inhibit expression of IL-6, IL-8 and other cytokinessuch as tissue necrosis factor (TNF-α). Inhibition of inflammatorycytokines IL-1 and TNF-α, by rhein and diacerein is described, forinstance, in WO 02/058681, WO 01/051044, J. Martel-Pelletier et al.Journal of Rheumatology, 1998, 25(4), 753-762, E. Douni et al. ArthritisRes Ther, 2004, 6: R65-R72.

Chondrocytes from patients suffering from these conditions express highlevels of TNF-α, and IL-1, compared to chondrocytes from healthyindividuals, and this specific mechanism of action of rhein as an IL-1inhibitor is believed to explain, at least in part, the effectiveness ofrhein and diacerein in the treatment of certain arthritic conditions,for instance, rheumatoid arthritis, osteoarthritis and psoriaticarthritis.

The metabolite Aloe-emodin present in diacerein has, however, been shownto have clastogenic effect in colon and kidney cells. Genotoxicity ofAloe-emodin, tested by Comet assay, is reported, for instance, by S. O.Müller et al., Mutation Research, 371, (1996), 165-173.

Further, rhein and diacerein have the drawback of being poorly solublein aqueous solution, making the preparation of pharmaceutical dosageforms, from which the therapeutic agent is bioavailable, difficult. Thepoor solubility of rhein and diacerein is a particular problem withrespect to formulations for parenteral administration.

Diacerein, and its active metabolite rhein, are known to have a tendencyto produce laxative effects in patients over long-term treatment. It isbelieved that this laxative effect may be attributed, at least in part,to the very poor solubility of rhein and diacerein.

There is an ongoing need to provide further compounds for the treatmentor therapy of conditions that are influenced or mediated bypro-inflammatory cytokines of the IL-1 family, particularly inflammatoryand auto-immune diseases, including arthritic diseases.

It would further be advantageous to provide alternative compounds havingactivity for the inhibition of pro-inflammatory cytokines of the IL-1family, and which allow to address certain drawbacks of rhein and/ordiacerein.

It would be further advantageous to provide compound having improvedactivity for the inhibition of pro-inflammatory cytokines of the IL-1family.

There are now provided novel pharmaceutical compounds of the formula(I):

wherein R₁, R₂, R₃ are each independently H or a C₁₋₄ alkyl group or aC₂₋₄ acyl group;R₄ and R₅ are each independently H or a group of formula —SO₃R₆, whereinR₆ is H or a C₁₋₄ alkyl group or a C₂₋₄ acyl group;with the proviso that at least one of R₄ and R₅ is a group of formula—SO₃R₆.

According to a particular embodiment of the present invention there isprovided a compound of the formula (I) wherein R₁, R₂, R₃ and R₄ are H,and R₅ is —SO₃H (formula (III)).

It has unexpectedly been found by the inventors that compounds of theformula (I) are capable of inhibiting production of pro-inflammatorycytokines of the IL-1 family. Compounds of formula (I) wherein R₄ and/orR₅ is —SO₃H have also been shown to exhibit advantageous solubilityproperties.

According to one aspect of the present invention there is provided aprocess for the preparation of a compound of formula (I), whichcomprises treating the corresponding compound of formula (II):

Wherein R₁, R₂ and R₃ are each independently H, with sulphuric acid.

According to one aspect of the invention there is now provided apharmaceutical composition comprising a compound of formula (I) incombination with suitable pharmaceutically acceptable excipients.

According to further aspects, the present invention relates to acompound of formula (I) for use as a medicament for human or veterinaryapplication, to a compound of formula (I) for the treatment ofconditions that are mediated or influenced by cytokines of the IL-1family, particularly for the treatment of inflammatory or auto-immunediseases, to the use of a compound of formula (I) for the preparation ofa medicament for the treatment of conditions that are mediated orinfluenced by cytokines of the IL-1 family, particularly inflammatory orauto-immune diseases, and to a method for the treatment of a conditionmediated or influenced by cytokines of the IL-1 family, which comprisesadministering to a subject a therapeutically effective amount of acompound of formula (I).

Other objects and advantages of the present invention will be apparentfrom the claims and the following detailed description, examples andaccompanying drawings.

FIGS. 1( a), 1(b) and 1(c) show the ¹H-NMR spectra of a compoundaccording to the present invention;

FIG. 2 shows the MS analysis of the same compound according to thepresent invention;

FIG. 3 is a graphical representation showing the effects of a compoundaccording to the present invention on the inhibition of IL-1β cytokineproduction in human chondrocytes; and

FIG. 4 is a graphical representation showing the dose-dependentinhibition of IL-1β cytokine production in human chondrocytes by acompound according to the present invention.

The present invention provides a compound of the formula (I):

Wherein R₁, R₂, R₃ are each independently H or a C₁₋₄ alkyl group or aC₂₋₄ acyl group;R₄ and R₅ are each independently H or a group of formula —SO₃R₆, whereinR₆ is H or a C₁₋₄ alkyl group or a C₂₋₄ acyl group;with the proviso that at least one of R₄ and R₅ is a group of formula—SO₃R₆.

According to one embodiment of the invention R₁ and R₂ are independentlyselected from H, a C₁₋₄ alkyl group or a C₂₋₄ acyl group; R₃ is H, andR₄ and R₅ may be H or —SO₃H, with the proviso that at least one of R₄and R₅ is —SO₃H. In a preferred embodiment, R₁ and R₂ are either both Hor both acetyl groups, R₃ and R₄ are both H, and R₅ is —SO₃H.

According to one embodiment of the invention R₁, R₂, R₃ are H, and R₄and R₅ are independently H or —SO₃H, with the proviso that at least oneof R₄ and R₅ is —SO₃H.

According to a preferred embodiment of the present invention there isprovided a compound of the formula (III)(6-sulfo-4,5-dihydroxy-9,10-dioxo-2-anthracene carboxylic acid):

The compounds of the present invention may be in the form of apharmaceutically acceptable salt thereof. Particularly sodium, potassiumor ammonium salts are contemplated.

Compounds of formula (I) may be prepared by a process according to thepresent invention, which comprises treating a compound of formula (II)

in which R₁, R₂ and R₃ are H, with concentrated sulphuric acid toproduce the corresponding compound of formula (I), in the form of thesulphonic acid. In a further step desired C₁₋₄ alkyl group or a C₂₋₄acyl group can be selectively substituted using conventional techniques.For instance, reaction with a C₂₋₄ acyl halide, or corresponding acylanhydride, to introduce the desired C₂₋₄ acyloxy group, reaction with aC₁₋₄ alcohol to form the corresponding ester, or for instance reactionwith diazomethane (CH₂N₂) to introduce C₁ alkyl group or with a C₂₋₄alkyl halide to introduce the corresponding C₂₋₄ alkyl group. Dependingon the desired substitution, known protecting groups may be introduced,where necessary, and cleaved using conventional processes.

Alternatively, sulphuric acid can be replaced with pyrosulphuric acid.

The reaction with sulphuric acid is preferably carried out at atemperature between 60 and 120° C., preferably around 100° C.

The reaction time with the acid may vary, dependent for instance on thereaction temperature, the acid used, the desired product (i.e. di- ormono-sulphonic acid substitution) etc. As a general indication reactiontimes of between 1 hour and 48 hours may be envisaged, for instancearound 24 hours. The progress of the reaction may advantageously bemonitored, for example by HPLC, and the reaction stopped at completionof the reaction to the desired di- or mono-sulphonate substitutedproduct.

The product may be isolated in the form of its corresponding salt, forinstance by addition of the corresponding metal halide (e.g. NaCl), orthe corresponding metal alkalizing agent (such as NaOH, KOH or NH₃).Salts envisaged include any pharmaceutically acceptable salt, such as,for instance, sodium, potassium or ammonium.

The thus obtained compound of formula (I) may be purified using anysuitable conventional purification process, such as, for instance,preparative HPLC or liquid-liquid partitioning.

Compounds of formula (I) according to the present invention exhibitactivity in the inhibition of pro-inflammatory cytokines of the IL-1family.

In in-vitro studies in human chondrocytes, compounds of formula (I) haveunexpectedly been shown to exhibit improved inhibition of interleukin-1(IL-1β), compared to rhein.

Advantageously, compounds of formula (I) can allow to avoid themetabolite aloe-emodin, present in diacerein.

In view of their activity in the inhibition of pro-inflammatorycytokines of the IL-1 family, compounds of the present invention arecontemplated for the treatment of conditions characterised by anabnormally high or increased level of IL-1.

The conditions that may be treated with compounds of the presentinvention include inflammatory and auto-immune diseases. Conditions thatmay be mentioned include rheumatoid arthritis, osteoarthritis,osteoporosis, psoriatic arthritis, psoriaris, artherosclerosis, Paget'sdisease, chronic heart disease, inflammatory bowl diseases includingulcerative colitis and Crohn's disease, endometriosis, Wegener'sgranulomatosis, neurological dysfunctions such as Alzheimer's diseaseand Parkinson's disease, myeloma, myeloid leukaemia, bone metastasis,diabetic nephrosis, pneumonary emphysema, asthma.

Accordingly, one aspect of the present invention relates to a method oftreating conditions characterised by increased levels of IL-1, ascompared to healthy individuals, which comprises administering to asubject an effective amount of a compound according to the presentinvention or a pharmaceutically acceptable salt thereof. The conditionsare preferably inflammatory diseases or auto-immune diseases.Particularly the conditions to be treated include inflammatory diseasesof the joints in particular osteoarthritis or rheumatoid arthritis.Psoriatic arthritis and psoriasis may also be particularly mentioned.

It is likely that the compounds of the present invention will be ofclinical utility in the wide range of inflammatory and auto-immunediseases described above, due also to their improved physical propertiescompared with rhein.

According to one aspect of the invention there is provided apharmaceutical composition comprising the compound of formula (I) incombination with suitable pharmaceutically acceptable excipients.Pharmaceutical compositions according to the present invention may befor human or veterinary use.

The pharmaceutical compositions according to the present invention mayhave a formulation suitable for administration by any route, including,for example, oral, intramuscular, intravenous, subcutaneous, rectal,topical, transcutaneous, intranasal, intrarticular, sublingual andintraperitoneal administration.

Formulations for oral administration may include, for instance, tablets,hard or soft gelatin capsules, lozenges, aqueous or oily suspensions,dispersible powders or granules for reconstitution, syrups or emulsions.

Formulations for parenteral administration may be in any suitablepharmaceutical form, such as in the form of a sterile injectable aqueousbuffered solution or suspension, as a sterile injectable solution orsuspension in any other non-toxic parenterally acceptable diluent orsolvent, or in a freeze-dried form for reconstitution at the time ofuse.

The compositions of the present invention may be also be provided informulations for topical administration, for instance in the form of acream, gel, ointment or emulsion in an aqueous or oily carrier.

Compounds of formula (I) according to the present invention are expectedto show better solubility in water than rhein or diacerein due to thepresence of the sulphonate group. For instance, compounds of formula (I)according to the present invention wherein R₁, R₂, R₃ are H and R₄ andR₅ are independently or both SO₃H, have been shown to exhibitparticularly good solubility in aqueous solution. For example, thecompound of formula (III) has a solubility in water of 1.2 mg/ml whilerhein and diacerein are practically insoluble in water.

The good solubility properties of the compounds of the present inventionallows the compounds to be advantageously administered by parenteralroutes, e.g. by injection or infusion, particularly as intraarticular,intramuscular, intravenous or subcutaneous injection or infusion.

The pharmaceutical compositions may be prepared according to methodsknown in the art, using suitable known pharmaceutically excipientsand/or additives.

Any suitable conventional pharmaceutically acceptable excipients arecontemplated, for example diluents, binders, surfactants, lubricants,suspending agents, emulsifiers, buffers anticaking agents, aqueous oroily carriers, disintegrating agents, preserving agents, flavouringagents, sweetening agents, colouring agents, in accordance with theselected route of administration.

Suitable dosage regimes will vary dependent, amongst other things, onfactors such as the therapeutic application, the severity of thecondition and with respect to the patient to be treated. Typical dailydosage amounts may vary in the order of from about 0.05 mg to about 150mg per kg of patient bodyweight per day. In general terms a daily dosageof from about 10 mg to 500 mg per day, such as between about 10 mg andabout 250 mg per day may be envisaged. The amount of active ingredientin a unit dosage from will depend on the above factors and also on thechosen route of administration, and will generally be in the region of 1mg to 500 mg of active ingredient per unit dosage form.

The invention is further illustrated by the following-non limitingexamples.

EXAMPLES Example 1 Preparation of6-sulfo-4,5-dihydroxy-9,10-dioxo-2-anthracene carboxylic acid

Rhein (1 g) produced by the deacetylation of pure diacerein, having apurity more than 99%, was dissolved in concentrated sulphuric acid (100ml). The solution was heated at 100° C. and stirred for 24 hours.Progress of the reaction was followed in real time using HPLC tocompletion of the reaction. The reaction mixture was then allowed tocool and poured into 21 water with stirring. The resultant solution wasthen stored over the night at 4° C. The unreacted rhein salting materialwas eliminated by centrifuge. The compound6-sulfo-4,5-dihydroxy-9,10-dioxo-2-anthracene carboxylic acid wasprecipitated as its sodium salt by the addition of 110 g of sodiumchloride. The resulting suspension was cooled at 4° C. for one hour,then centrifuged to separate out the solid product and dried undervacuum. 2.32 g of product was obtained.

Elimination of Salts:

The product obtained above was added to water (115 ml) with mixing at 4°C. for 30 minutes. The suspension was then centrifuged and decanted toeliminate the supernatant with the residual salts. This operation wasrepeated seven times until a constant conductivity (around 330 μS/cm),measured with a conductivity meter (Radiometer CDM 206). The residueafter centrifuging was then dried under vacuum to obtain 480 mg ofproduct, with a purity of 95.6% as determined by HPLC.

Example 2 Characterization ¹H-NMR Analysis:

¹H-NMR spectrum of the product obtained in example 1 was carried out indimethyl sulphoxide (DMSO) using a Bruker® spectrometer at 400 MHz. Thespectra obtained, shown in FIGS. 1( a) to 1(c), shows concordance withthe product 6-sulfo-4,5-dihydroxy-9,10-dioxo-2-anthracene carboxylicacid.

The ¹H-NMR spectra indicates the presence of only four aromatic protons,two in the meta position and two in ortho positions. Showing that thesulphonic acid substitution has occurred in the ring which previouslyhad only a hydroxyl substituent. The chemical displacements (shifts),compared to calculated chemical displacements on the basis ofincrements, show the substitution of the sulphonic acid group to be inthe ortho position.

MS Analysis:

Mass spectroscopy carried out on the product of example 1 with anAgilent 1100 LC-MS Spectrometer, with ionisation at atmospheric pressurein negative electro-spray mode. The spectrum obtained, shown in FIG. 2,shows a peak at 364, and thus concords with the product6-sulfo-4,5-dihydroxy-9,10-dioxo-2-anthracene carboxylic acid, formula(III).

Example 3 Preparation of 6-sulfo-4,5-dihydroxy-9,10-dioxo-2-anthracenecarboxylic acid sodium salt

Rhein (1 g) produced by the deacetylation of pure diacerein, having apurity more than 99%, was dissolved in concentrated sulphuric acid (100ml). The solution was heated at 100° C. and stirred for 24 hours.Progress of the reaction was followed in real time using HPLC tocompletion of the reaction. The reaction mixture was then allowed tocool and poured into 21 water with stirring. The resultant solution wasthen stored over the night at 4° C. The unreacted rhein salting materialwas eliminated by centrifuge. The pH of the surnatant was adjusted to7.0 with NaOH 1M. The product6-sulfo-4,5-dihydroxy-9,10-dioxo-2-anthracene carboxylic acid, in theform of its sodium salt, was then isolated and purified from thesolution by preparative HPLC using a reversed phase C18 silica columnand methanol/water/H₃PO₄ eluent. 1 g of product was isolated andcharacterised as 6-sulfo-4,5-dihydroxy-9,10-dioxo-2-anthracenecarboxylic acid sodium salt by NMR and MS as in example 2.

Example 4 In Vitro Studies on IL-13 Inhibition in Human Chondrocytes

The activity of the compound of example 1, and of rhein in inhibitingthe lipopolysaccharide (LPS) stimulated production of IL-1β cytokine byhuman normal and osteoarthritic (OA) chondrocytes was studied.

Materials and Methods:

Human cartilage was obtained during orthopaedic surgery for total hipprosthesis due to traumatic fracture in normal subjects or OA patients.Patients with OA were selected on the basis of the following criteria(1) bi-lateral OA, (2) diagnoses of moderate OA (grade I-III,Kellgren-Laurence), supported by radiology and pathology.

A suspension of isolated chondrocytes was prepared from cartilagespecimens obtained from four OA patient and nine normal subjects.Specimens were obtained and maintained under aseptic conditions. Thecartilage was cut into small fragments and incubated with 1 mg/mlclostridial collagenase in carbonate/bicarbonate buffer for 48 hours at37° C. Once separated from the cartilage matrix, chondrocytes werecentrifuged at 1500 RPM for five minutes. The separated chondrocytes,suspended in culture medium (DMEM supplemented with 10% SCF) were usedfor the experiment. Cell viability was evaluated by Trypan blueexclusion.

In vitro stimulation of chondrocytes with bacterial endotoxin (LPS) forthe production of IL-1β was carried out as follows:

Aliquots of 1×10⁶ cells/ml in culture medium were seeded in 15 ml Falcontubes and maintained under agitation on a gyratory shaker (100 RPM).

Chondrocytes were cultured for 48 hours in the presence of MPS (10μg/ml) with 20 mg/ml rhein, or increasing concentrations (1, 5, 10, 20and 30 μg/ml) of the test compound (compound of Example 1). For thesamples with rhein the solution was subjected to five minutes sonicationin order to minimize solubility problems related to the hydrophobicnature of rhein. This problem was not present for the solutions of thetest compound due to its good solubility.

IL-1β production by chondrocytes was assayed in the culture medium inthe various sample cultures by an ELISA kit.

FIG. 3 shows the results obtained with rhein and the compound of Example1 on IL-1β production in LPS-stimulated normal and OA chondrocytes, asevaluated by ELISA. FIG. 4 shows the dose-dependent inhibition of IL-1production of normal chondrocytes by the test compound (compound ofExample 1). The results illustrated in FIG. 3 show the compound of thepresent invention to exhibit significantly increased IL-1β inhibitionactivity compared with rhein. FIG. 4 shows the compound according to theinvention to provide significant inhibition of IL-1β production, withmaximum inhibition between 10 and 20 mg/ml.

1-17. (canceled)
 18. A method for the treatment of a disease mediated orinfluenced by pro-inflammatory cytokines of the IL-1 family, saiddisease being selected from neurological dysfunctions such asAlzheimer's disease and Parkinson's disease, osteoporosis,atherosclerosis, Paget's disease, chronic heart disease, inflammatorybowel diseases including ulcerative colitis and Crohn's disease,endometriosis, Wegener's granulomatosis, myeloma, myeloid leukaemia,bone metastasis, diabetic nephrosis, pneumonary emphysema, or asthma,which comprises administering to a subject a therapeutically effectiveamount of a compound of the formula (I):

wherein R₁, R₂, R₃ are each independently H or a C₁₋₄ alkyl group or aC₂₋₄ acyl group; R₄ and R₅ are each independently H or a group offormula —SO₃R₆, wherein R₆ is H or a C₁₋₄ alkyl group or a C₂₋₄ acylgroup; with the proviso that at least one of R₄ and R₅ is a group offormula —SO₃R₆, or a pharmaceutically acceptable salt thereof.
 19. Amethod for the treatment of a disease mediated or influenced bypro-inflammatory cytokines of the IL-1 family, said disease beingselected from Alzheimer's disease and Parkinson's disease, osteoporosis,atherosclerosis, Paget's disease, chronic heart disease, inflammatorybowel diseases including ulcerative colitis and Crohn's disease,endometriosis, Wegener's granulomatosis, myeloma, myeloid leukaemia,bone metastasis, diabetic nephrosis, pneumonary emphysema, or asthma,which comprises administering to a subject a therapeutically effectiveamount of a compound of the formula (I):

wherein R₁, R₂ are independently H or a C₁₋₄ alkyl group or a C₂₋₄ acylgroup, R₃ and R₄ are H, and R₅ is —SO₃H, or a pharmaceuticallyacceptable salt thereof.
 20. A method for the treatment of a diseasemediated or influenced by pro-inflammatory cytokines of the IL-1 family,said disease being selected from Alzheimer's disease and Parkinson'sdisease, osteoporosis, atherosclerosis, Paget's disease, chronic heartdisease, inflammatory bowel diseases including ulcerative colitis andCrohn's disease, endometriosis, Wegener's granulomatosis, myeloma,myeloid leukaemia, bone metastasis, diabetic nephrosis, pneumonaryemphysema, or asthma, which comprises administering to a subject atherapeutically effective amount of a compound of the formula (III):

or a pharmaceutically acceptable salt thereof.